e 64  (Tocris)

Journal: Cell Death Discovery

Article Title: The monkeypox virus suppresses autophagy by modulating Rubicon expression

doi: 10.1038/s41420-025-02920-z

Figure Lengend Snippet: A Calu-3 cells were infected with MPXV at an MOI of 0.5 or 1 for 24 and 48 h. Two hours before lysis, cells were treated with the lysosome inhibitors E64d/Pep.A as indicated (+). LC3-II and p62 levels were analyzed to monitor autophagy flux, while L1R levels were analyzed to control MPXV replication by western blot. Actin and Tubulin were included as a loading control. The graphs represent mean ± SEM of LC3-II:Actin, p62:Actin and L1R:Tubulin values from at least three independent experiments. B Calu-3 cells were infected with MPXV at MOI of 0.5 (represented in red) or 1 (represented in black) for 24, 48 h, and untreated (NT) or treated with E64d/Pep.A for 2 h. Kinetic of viral yield inside the cells (Cells) (expressed as Log copies/ng) and in supernatants (Sup) (expressed as Log copies/mL) were quantified by qRT-PCR. Experiments were performed as three independent replicates; mean ± SEM are shown in the picture.

Article Snippet: To block lysosomal activity, cells were treated with E64d and Pepstatin A (5 μg/mL) (Santa Cruz Biotechnology, sc-201280A, sc-45036) for 2 h before lysis.

Techniques: Infection, Lysis, Control, Western Blot, Quantitative RT-PCR

Journal: Cell Death Discovery

Article Title: The monkeypox virus suppresses autophagy by modulating Rubicon expression

doi: 10.1038/s41420-025-02920-z

Figure Lengend Snippet: A Calu-3 cells were infected with MPXV at an MOI of 0.5 or 1 for 24 h and 48 h. Two hours before lysis, cells were incubated with E64d/Pep.A as indicated (+). Rubicon and L1R levels were analyzed by western blot. HSP90 and Tubulin were included as loading controls. The graphs represent mean ± SEM of Rubicon:HSP90 values from three independent experiments. B Real-time PCR analysis of Rubicon mRNA levels in Calu-3 cells infected with MPXV at MOI of 0.5 or 1 for 24 or 48 h. Expression levels were normalized based on HSP90 values. A.U. arbitrary unit. Graph reports mean ± SEM of values from 3 independent experiments. C Calu-3 cells were treated for 4 h with the proteasomal inhibitor MG132 at a final concentration of 10 or 5 µM. Rubicon levels were analyzed by western blot. Actin was included as a loading control. The graphs represent mean ± SEM of Rubicon:Actin values from three independent experiments. D Calu-3 cells were treated for 4 h with the proteasomal inhibitor MG132 at a final concentration of 5 µM or infected with MPXV at MOI of 0.5 48 h. p21, p53, K48-linked ubiquitin (ubq) chains, and Rubicon levels were analyzed by western blot. L1R levels were monitored to verify MPXV replication. Actin and Tubulin were included as loading controls. The graphs represent mean ± SEM of p21:Actin, p53:Actin, L1R:Actin K48-Ubiquitin:Tubulin, Rubicon:Actin values from three independent experiments.

Article Snippet: To block lysosomal activity, cells were treated with E64d and Pepstatin A (5 μg/mL) (Santa Cruz Biotechnology, sc-201280A, sc-45036) for 2 h before lysis.

Techniques: Infection, Lysis, Incubation, Western Blot, Real-time Polymerase Chain Reaction, Expressing, Concentration Assay, Control, Ubiquitin Proteomics

Journal: Cell Death Discovery

Article Title: The monkeypox virus suppresses autophagy by modulating Rubicon expression

doi: 10.1038/s41420-025-02920-z

Figure Lengend Snippet: Calu-3 cells silenced for Rubicon gene expression (iRUBCN#1, iRUBCN#2) or not (iCtr), were infected at an MOI of 0.5 and cultured for 48 h. Two hours before lysis, cells were incubated with E64d/Pep.A as indicated. A LC3-II levels were analyzed to monitor autophagy flux by western blot. Actin was included as a loading control. The graph represents mean ± SEM of LC3-II:Actin values from three independent experiments. B Rubicon levels were analyzed by western blot. Tubulin was included as a loading control. The graphs represent means ± SEM of Rubicon:Tubulin values from three independent experiments. C L1R levels were analyzed to verify MPXV replication by western blot. Actin was included as a loading control. The graphs represent mean ± SEM of L1R:Actin values from three independent experiments. D Calu-3 cells were silenced for Rubicon gene expression: iRUBCN#1 (represented in black), iRUBCN#2 (represented in purple), or not (iCtr, represented in blue), infected at an MOI of 0.5 and cultured for 48 h. Kinetics of viral yield inside the cells (Cells) (expressed as copies/ng) and in supernatants (Sup) (expressed as copies/mL) were quantified by qRT-PCR. E Viral titer was evaluated as Log TCID50/mL in supernatants of Calu-3 infected cells with MPXV at MOI of 0.5 for 48 h, and transfected either with iRUBCN#1 (represented in black) or iRUBCN#2 (represented in purple) and iCtr (represented in blue). Experiments were performed as three independent replicates; mean ± SEM are shown in the picture.

Article Snippet: To block lysosomal activity, cells were treated with E64d and Pepstatin A (5 μg/mL) (Santa Cruz Biotechnology, sc-201280A, sc-45036) for 2 h before lysis.

Techniques: Gene Expression, Infection, Cell Culture, Lysis, Incubation, Western Blot, Control, Quantitative RT-PCR, Transfection